Tumour necrosis factor alpha (TNFα) is a cytokine produced by many cell types, mainly activated monocytes and macrophages. It is expressed as a 26 kDa integral transmembrane precursor protein from which a mature protein of approximately 17 kDa is released by proteolytic cleavage. The soluble bioactive TNFα is a homotrimer that binds cell surface receptors. TNFα has been shown to induce necrosis of solid tumours. It exerts its effects mainly on the endothelium of the tumour-associated vasculature, with increased permeability, upregulation of tissue factor, fibrin deposition and thrombosis, and massive destruction of the endothelial cells.
Interleukin-2 (IL2), a four a helix bundle cytokine produced by T helper 1 cells, plays an essential role in the activation phases of both adaptive and innate immune responses. Although it is not believed to have a direct cytotoxic effect on cancer cells, it has been reported to induce tumour regression by stimulating a cell-mediated immune response.
Intratumoural injections of IL2 have been trialled in metastatic melanoma patients [1]. In that study, treatment was administered three times weekly for at least 2 weeks, and overall 69% of patients were reported to achieve a complete response.
WO01/66298 described immunoconjugates comprising TNFα and IL2 respectively, fused to antibody L19. L19 specifically binds the ED-B domain of fibronectin isoform B-FN, which is one of the best known markers angiogenesis (U.S. Ser. No. 10/382,107; WO01/62298). ED-B is an extra domain of 91 amino acids found in the B-FN isoform and is identical in mouse, rat, rabbit, dog and man. B-FN accumulates around neovascular structures in aggressive tumours and other tissues undergoing angiogenesis, such as the endometrium in the proliferative phase and some ocular structures in pathological conditions, but is otherwise undetectable in normal adult tissues.
Carnemolla et al. [2] described enhancement of the antitumour properties of IL2 by its targeted delivery to the tumour blood vessel extracellular matrix in an L19-IL2 immunoconjugate.
Christ et al. [3] described intratumoural administration of an IL2 immunoconjugate, a TNFα immunoconjugate, or antibody alone. The antibody used was anti-EGFR, which had an anti-tumour effect. An anti-tumour immune response was reported following multiple injections of either fusion protein.
Borsi et al. [4] reported a study in which L19-TNFα and L19-IL2 immunoconjugates were administered intravenously to mice on days 7 and 10 following implantation of tumour cells. L19 was used to concentrate and maximise the anti-tumour effects of the systemically delivered cytokines. The combination of immunocytokines was reported to have a synergistic effect on tumour volume. Mice who received the combination treatment had markedly reduced tumour volume compared with those who received just one immunocytokine.